Sham-burned Animals Research Articles

224 Burn Injury Induced Functional and Morphological Alterations in Skeletal Mitochondria in a Metabolically Characterized Animal Model

Altered mitochondrial biology and function is considered as an underlying mechanism of burn injury induced hypermetabolism and insulin resistance. Our previous work has characterized a burn mouse model with insulin resistance by in vivo clamp method accompanied with muscle insulin signal proteins in skeletal muscle. The present study aimed at exploring the morphology and functional changes of muscle mitochondrial in this characterized burn animal model. C57BL/6 mice (10–12 weeks) received 30% total body surface area (TBSA) full thickness burn injury or sham burn injury. Gastrocnemius and quadriceps muscles were collected on days 1, 3, and 7 after burn injury. Mitochondrial function was assessed by citrate synthase activity, mitochondrial membrane potential and NAD+/NADH ratio. Gene expression related to mitochondrial biogenesis, dynamics and mitochondrial DNA contents were quantified with real time quantitative PCR. The morphological changes were assessed by a quantitative 2-dimensional transmission electron microscopy (TEM). Our studies demonstrated that the major changes occurred on day 3. Mitochondrial dysfunction was revealed with significantly reduced citrate synthase activity (fig A, B, C), mitochondrial membrane potential and NAD+/NADH ratio. This was accompanied with reduced mitochondrial DNA contents and mRNA expression of cytochrome C (CytC), mitochondrial transcription factor A (TFAM), nuclear respiratory factor 1 (NRF1) and fission protein 1(Fis1) (fig D, E). TEM demonstrated that mitochondria became longer and branched with disrupted cristae as compared to sham burn animals (fig F, G). There was a time dependent reduction to number of cristae time-dependently (fig H). Since CytC is the major component of electron transport chain, NRF1 regulates transcription rregulating cellular growth and nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication, the latter is also regulated by TFAM and Fis1 is one of the mitochondrial fission factors. We conclude that the above molecular and morphological changes mitochondria are part of the mechanisms of metabolic aberrations caused by burn injury.

Effect of Fasting on the Metabolic Response of Liver to Experimental Burn Injury

Liver metabolism is altered after systemic injuries such as burns and trauma. These changes have been elucidated in rat models of experimental burn injury where the liver was isolated and perfused ex vivo. Because these studies were performed in fasted animals to deplete glycogen stores, thus simplifying quantification of gluconeogenesis, these observations reflect the combined impact of fasting and injury on liver metabolism. Herein we asked whether the metabolic response to experimental burn injury is different in fed vs. fasted animals. Rats were subjected to a cutaneous burn covering 20% of the total body surface area, or to similar procedures without administering the burn, hence a sham-burn. Half of the animals in the burn and sham-burn groups were fasted starting on postburn day 3, and the others allowed to continue ad libitum. On postburn day 4, livers were isolated and perfused for 1 hour in physiological medium supplemented with 10% hematocrit red blood cells. The uptake/release rates of major carbon and nitrogen sources, oxygen, and carbon dioxide were measured during the perfusion and the data fed into a mass balance model to estimate intracellular fluxes. The data show that in fed animals, injury increased glucose output mainly from glycogen breakdown and minimally impacted amino acid metabolism. In fasted animals, injury did not increase glucose output but increased urea production and the uptake of several amino acids, namely glutamine, arginine, glycine, and methionine. Furthermore, sham-burn animals responded to fasting by triggering gluconeogenesis from lactate; however, in burned animals the preferred gluconeogenic substrate was amino acids. Taken together, these results suggest that the fed state prevents the burn-induced increase in hepatic amino acid utilization for gluconeogenesis. The role of glycogen stores and means to increase and/or maintain internal sources of glucose to prevent increased hepatic amino acid utilization warrant further studies.

In situ metabolic flux analysis to quantify the liver metabolic response to experimental burn injury

Trauma such as burns induces a hypermetabolic response associated with altered central carbon and nitrogen metabolism. The liver plays a key role in these metabolic changes; however, studies to date have evaluated the metabolic state of liver using ex vivo perfusions or isotope labeling techniques targeted to specific pathways. Herein, we developed a unique mass balance approach to characterize the metabolic state of the liver in situ, and used it to quantify the metabolic changes to experimental burn injury in rats. Rats received a sham (control uninjured), 20% or 40% total body surface area (TBSA) scald burn, and were allowed to develop a hypermetabolic response. One day prior to evaluation, all animals were fasted to deplete glycogen stores. Four days post-burn, blood flow rates in major vessels of the liver were measured, and blood samples harvested. We combined measurements of metabolite concentrations and flow rates in the major vessels entering and leaving the liver with a steady-state mass balance model to generate a quantitative picture of the metabolic state of liver. The main findings were: (1) Sham-burned animals exhibited a gluconeogenic pattern, consistent with the fasted state; (2) the 20% TBSA burn inhibited gluconeogenesis and exhibited glycolytic-like features with very few other significant changes; (3) the 40% TBSA burn, by contrast, further enhanced gluconeogenesis and also increased amino acid extraction, urea cycle reactions, and several reactions involved in oxidative phosphorylation. These results suggest that increasing the severity of injury does not lead to a simple dose-dependent metabolic response, but rather leads to qualitatively different responses.

Preliminary study of skeletal muscle apoptosis after severe thermal injury in rats

To investigate changes in skeletal muscle apoptosis after a severe thermal injury in rats. One hundred Wistar rats were randomly divided into two groups: sham thermal injury group and severe thermal injury group. They were subdivided into 1, 4, 7, 10, 14 days post-injury with 10 rats in each subgroup. Rats in severe thermal injury group were subjected to a 40% total body surface area full-thickness scald injury. Both weight and tibialis anterior (TA) mass of rats were weighed on 1, 4, 7, 10, 14 days post-injury. Electron microscope was used for observing ultrastructural changes in skeletal muscle, including apoptosis. Tissues of tibialis anterior from burn and sham burn animals were then examined by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry. Compared with sham thermal injury group, body weight and TA mass of rats were decreased from first day on, and it dropped to the lowest level at 4 days (P<0.05 and P<0.01), and started to regain from 7 days on (all P<0.01). Electron micrographs showed condensation of chromatin around the periphery of the nucleus, blebbing of the sarcolemma, and free of myofibrils near myonuclei in a large area in skeletal muscle of thermally injured rats. Sporadic TUNEL positive myonuclei were also seen under light microscope in skeletal muscle in thermal injury group. There were no characteristic signs of apoptosis in skeletal muscle in rats of sham group. There are skeletal muscle apoptosis after severe thermal injury. It may contribute to atrophy of skeletal muscle after burn injury in rats.

Renin‐Angiotensin System Blockade Improves Skeletal Muscle Glucose Uptake In Burn Injury

Insulin resistance after burn is associated with alterations in postreceptor insulin signaling and abnormal glucose homeostasis. We have previously shown that renin‐angiotensin system blockade using losartan, an AT1 receptor blocker, improves insulin signaling and glucose tolerance in burn injured rats. In this study, we examined the effects of losartan on 2‐deoxy‐glucose uptake into the soleus muscle of burned rats. A 30% body surface area burn was induced by immersion of the dorsum into 90ºC water for 15s. Sham burned rats were immersed in 23ºC water. Losartan (30 mg/kg/day) or placebo (water) was given by gavage for three days post‐burn resulting in sham burn (n= 8), burn placebo (n= 8) burn losartan (n= 8). Insulin (100 nM) stimulated glucose uptake was impaired in the soleus muscle of burn placebo animals as determined by 2‐deoxy‐glucose uptake, in vitro (77% decrease compared with sham). However, losartan treatment completely reversed this effect returning the uptake to the level of the sham burn animals. This suggests that the renin‐angiotensin system is involved in the insulin resistance of skeletal muscle that occurs in burn injury.

INCREASING PERCENT BURN IS CORRELATED WITH INCREASING INFLAMMATION IN AN ADULT RODENT MODEL

Burn injury has been associated with systemic/compartmental inflammatory responses and myocardial dysfunction. We hypothesized that burn size correlates with the extent of cardiac inflammatory response/contractile dysfunction. Adult male Sprague-Dawley rats were divided to receive anesthesia, a 3-degree burn covering 20%, 30%, 40%, or 60% total body surface area (TBSA) plus fluid resuscitation (lactated Ringer, 4 mL/kg per percent burn); sham burn animals were included as controls. There were seven rats in each group. Rats were euthanized Twenty-four h postburn, and TNF-alpha, IL-1beta, and IL-6 were measured in the plasma and in supernatant from isolated cardiac myocytes by enzyme-linked immunosorbent assay. In addition, left ventricular function (Langendorff) was studied in vitro, and troponin levels were measured by enzyme-linked immunosorbent assay. There were progressive, statistically significant increases in plasma and myocyte inflammatory cytokine levels, as well as plasma troponin with increasing burn size. Similarly, left ventricular pressure (in millimeters of mercury) and +/-dP/dtmax (in millimeters of mercury per second) progressively fell with increasing burn size. However, myocardial contractile depression induced by 60% TBSA burn was similar to that produced by 40% TBSA burn. These data suggest that the degree of inflammatory response, cardiac tissue injury, and myocardial contractile depression were correlated directly with the percent TBSA burn. However, unlike inflammation and cardiac tissue damage, myocardial contractile depression reached a plateau, with maximal myocardial contraction and relaxation defects observed at 40% TBSA burn, which were not further aggravated by a larger (60%) burn.

Interleukin-6, TNF-alpha and interleukin-1 beta levels in blood and tissue in severely burned rats.

Previous studies have demonstrated the early appearance of inflammatory cytokines in the systemic circulation after thermal injury both in humans and animals. The aim of this study was to evaluate the time course of several cytokines, IL-6, TNF-alpha and IL-1beta in serum, lung, liver and brain of severely burned rats during the first week after thermal injury. Cytokine measurements were performed by enzyme-linked immunosorbent assay (ELISA). The comparison between the sham-burned animals and animals with third-degree burns on 20% or 40% of their total body surface area allowed for the study of the inflammatory process relative to the size of the injury. Serum IL-6 levels, which were undetectable in sham-treated animals, peaked during the first hours after injury and were proportionate to the size of the area burned. After a few days, IL-6 increased once more, but only in the most severely burned rats. In lung, liver and brain, low but measurable basal levels of TNF-alpha and IL-1 were detected in sham-burned animals. Strikingly, IL-1beta levels remained significantly elevated in the lung after injury in animals having 20% and 40% burned skin area. Unexpectedly, both TNF-alpha and IL-1beta production decreased gradually in liver and brain after burn injury. Also, the inflammatory response after a burn injury appeared to be biphasic. The first period corresponded to the early release of IL-6 into the circulation, proportional to the severity of the injury. After a few days, a second period was marked by the extension of the inflammatory processes from the injured area to the rest of the body, particularly to lung, which could be considered as at potential risk of involvement in severely burned patients.

Dual effects of mesenteric lymph isolated from rats with burn injury on contractile function in rat ventricular myocytes

Gut-derived factors in intestinal lymph have been shown to trigger myocardial contractile dysfunction. However, the underlying cellular mechanisms remain unclear. We examined the effects of physiologically relevant concentrations of mesenteric lymph collected from rats with 40% burn injury (burn lymph) on excitation-contraction coupling in rat ventricular myocytes. Burn lymph (0.1-5%), but not control mesenteric lymph from sham-burn animals, induced dual positive and negative inotropic effects depending on the concentrations used. At lower concentrations (<0.5%), burn lymph increased the amplitude of myocyte contraction (1.6 +/- 0.3-fold; n = 12). At higher concentrations (>0.5%), burn lymph initially enhanced myocyte contraction, which was followed by a block of contraction. These effects were partially reversible on washout. The initial positive inotropic effect was associated with a prolongation of action potential duration (measured at 90% repolarization, 2.5 +/- 0.6-fold; n = 10), leading to significant increases in the net Ca2+ influx (1.7 +/- 0.1-fold; n = 8). There were no significant changes in the resting membrane potential. The negative inotropic effect was accompanied by a decrease in the action potential plateau (overshoot decrease by 69 +/- 10%; n = 4) and membrane depolarization. Voltage-clamp experiments revealed that the positive inotropic effects of burn lymph were due to an inhibition of the transient outward K+ currents that prolong action potential duration, and the inhibitory effects were due to a concentration-dependent inhibition of Ca2+ currents that lead to a reduction of action potential plateau. These burn lymph-induced changes in cardiac myocyte Ca2+ handling can contribute to burn-induced contractile dysfunction and ultimately to heart failure.

Decreased lymphocyte apoptosis by anti-tumor necrosis factor antibody in Peyer's patches after severe burn.

Severe burn results in immunosuppression, with increased lymphocyte apoptosis in both the central and peripheral immune system. As atrophy of the small intestine has been described in mouse models and intestinal lymphocytes have been implicated in the burn inflammatory response, we examined the effects of burn and tumor necrosis factor (TNF)-alpha on lymphocytes in intestinal Peyer's patches. Anesthetized C57BL6 mice received a 30% full-thickness scald burn on the upper back. Sham-burned animals served as controls. Anti-TNF or control immunoglobulin (Ig) G antibody (200 microg) was given immediately after the burn. The animals were initially resuscitated with 2 mL of normal saline, and were then sacrificed 12 h postburn. Terminal deoxyuridine nick-end labeling (TUNEL) and proliferative cell nuclear antigen (PCNA) staining was performed. Apoptosis was quantified as apoptotic lymphocytes/high-powered field (hpf). Results, expressed as mean +/- SEM, were compared using analysis of variance (ANOVA) and the Student-Newman-Keuls test. All mice survived the burn. An initial time-course experiment demonstrated maximal Peyer's patch apoptosis 12 h after the burn. Sham mice had 25 +/- 7 TUNEL-stained cells/hpf in Peyer's patches, whereas burned mice had 93 +/- 18 cells/hpf (P < 0.05). In contrast, burned mice receiving anti-TNF antibody had 28 +/- 8 TUNEL-stained cells/hpf (P < 0.05 vs. burn), whereas sham mice receiving anti-TNF antibody had 20 +/- 4 cells/hpf. There were no significant differences in PCNA staining between the groups. Scald burn results in lymphocyte apoptosis in Peyer's patches. This apoptosis can be abrogated by the addition of anti-TNF antibody. Apoptotic changes may lead to the failure of the intestinal immunological barrier and increased risk of sepsis.

IL-1beta and IL-6 act synergistically with TNF-alpha to alter cardiac contractile function after burn trauma.

Although numerous studies have provided evidence that the inflammatory cytokines TNF-alpha and IL-1beta have significant negative inotropic effects, the role of the interleukins in burn-mediated cardiac dysfunction has not been defined. Furthermore, most studies examining the cardiotoxic effects of inflammatory cytokines have ignored the complex inflammatory milieu that occurs in the intact subject with trauma, sepsis, or ischemic heart disease. Therefore, this study examined the time course of IL-1beta and IL-6 secretion by cardiomyocytes after burn trauma, and additional studies examined the effects of these cytokines alone or in combination with TNF-alpha on cardiac contractile performance (Langendorff). Sprague-Dawley rats were given a full thickness burn injury over 40% of the total body surface area; fluid resuscitation was lactated Ringers solution, 4 mL/kg per burn percentage of burn area. Sham burn animals received identical anesthesia and handling, but no burn injury. Rats were sacrificed at several different times postburn, and isolated hearts (n = 4-5 rats/group/time period) were perfused with collagenase-containing buffer to prepare cardiomyocytes or were perfused in vitro to examine cardiac contractile function (n = 5-6 rats/group/time period). Additional naive control rats (n = 10) were included to prepare cardiomyocytes that, in turn, were challenged with different concentrations of either IL-1beta, IL-6, or TNF-alpha alone or in combination for several time periods (CO2 incubator at 37 degrees C for 1-3 h). Finally, inflammatory cytokines alone or in combination were added to the perfusate of hearts isolated from additional control rats (n = 6-7/group) to assess the cardiac contraction and relaxation effects of cytokine challenge. Despite aggressive fluid resuscitation, burn trauma produced a time-related increase in cardiomyocyte secretion of IL-1beta, IL-6, and TNF-alpha. Exposure of naive cardiomyocytes prepared from control rats to each cytokine alone or combined cytokine challenge produced a time-dependent and concentration-dependent decrease in cell viability and an increase in supernatant creatine kinase levels. Either IL-1beta or TNF-alpha produced greater cardiac defects than IL-6 when added separately to Langendorff-perfused hearts; dysfunction was maximal with combined cytokine challenge (IL-1beta plus TNF-alpha plus IL-6). The data confirm that burn trauma upregulates inflammatory cytokine secretion by cardiomyocytes and suggest that these inflammatory cytokines act in concert to produce burn-mediated cardiac contractile dysfunction.

Injury induces deficient interleukin-12 production, but interleukin-12 therapy after injury restores resistance to infection.

To assess at serial intervals the production of interleukin-12 (IL-12) by monocytes/macrophages from the peripheral blood of injured patients and control subjects, and using a mouse model to confirm human findings and explore the effectiveness of low-dose IL-12 therapy in restoring resistance to infection after injury. Serious injury is associated with loss of function of the T helper 1 lymphocyte phenotype, but little is known about IL-12 production in injured patients. The authors previously reported that early, moderate-dose IL-12 therapy in a mouse model of burn injury restored resistance to a later infectious challenge (cecal ligation and puncture, CLP). However, the efficacy of clinically relevant low-dose IL-12 therapy carried out to or beyond the time of septic challenge remains to be tested. Peripheral blood mononuclear cells (PBMCs) and adherent cells were obtained from 27 patients with major burns or traumatic injury and 18 healthy persons and were studied at serial intervals for IL-12 production stimulated by bacterial lipopolysacharide (LPS). PBMCs from 18 of the same patients were studied for IL-10 production as well. IL-12 production by adherent cells from the spleens of burn or sham burn mice was studied at serial intervals after injury to confirm the human findings. Low-dose IL-12 or vehicle was given every other day to groups of burn and sham burn mice, which were then challenged with CLP on day 10, and survival was determined. Finally, spleens were harvested from burn or sham burn animals receiving low-dose IL-12 or vehicle after CLP. After splenic cellularity was determined by hemocytometer, splenocytes were cultured and production of tumor necrosis factor-alpha, interferon-gamma, and IL-10 were assessed by immunoassay. Adherent cells from patients' PBMCs produced significantly less IL-12 than normal PBMCs after injury, reaching a nadir 8 to 14 days after injury. Stimulation of whole PBMCs by LPS indicated that at 8 to 14 days after injury, IL-12 production by PBMCs was significantly lower and IL-10 production was significantly higher than that of PBMCs from healthy persons. Low-dose IL-12 therapy significantly increased survival after CLP. Splenocytes from burn mice treated with IL-12 had significantly increased production of TNF-alpha and IF-beta, both before and after CLP, when compared with vehicle-treated burn animals. IL-10 production by bum splenocytes remained high after IL-12 treatment. Splenic cellularity increased after IL-12 treatment in burn mice. The capacity to produce IL-12 by adherent cells of the monocyte/macrophage lineage is significantly reduced after serious injury in humans and in a mouse burn model. In humans, there is a reciprocal relation between diminished IL-12 production and increased IL-10 production at approximately 1 week after injury. Low-dose IL-12 therapy in the mouse burn model markedly increased survival after a septic challenge, even when treatment was carried beyond the onset of sepsis. Low-dose IL-12 treatment in the mouse increased production of proinflammatory mediators important in host defense and at the same time maintained or increased production of IL-10, an important antiinflammatory cytokine.

Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis.

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43+/-1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringer's solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and +/-dP/dt(max). Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

Acetylcholine-Induced and Nitric Oxide-Mediated Vasodilation in Burns

Microvascular endothelial cells actively participate in local regulation of blood flow and blood–tissue exchange by producing various vasoactive substances including nitric oxide (NO). This study examined microcirculatory changes in the early stage of thermal injury and the NO-related mechanisms. Resistance arterioles of rat cremaster muscle were observed using intravital microscopy. Arteriolar diameter and flow velocity were measured and flow rate was calculated after administration of various vasoactive agonists in burns. In fluid-resuscitated rats with stable systemic blood pressure, microvascular caliber and blood flow were not significantly altered in the first hour following a 25% total body surface area full-thickness scald burn. Topical application of acetylcholine (ACh), an endothelium-dependent vasodilator, increased arteriolar diameter and flow rate in a dose-dependent fashion. The dose–responsive effects of ACh were significantly greater in burned rats than in sham-burned rats, and the augmentation was blocked by inhibition of NO production withNG-monomethyl-l-arginine (L-NMMA). Topical application of adenosine, an endothelium-independent vasodilator, and sodium nitroprusside, an exogenous NO donor, markedly increased arteriolar diameter and flow rate. The effects were not significantly different in burned and sham-burned animals, and the adenosine-induced vasodilation was not blocked by L-NMMA. These data suggest that endothelium-dependent and NO-mediated arteriolar dilation is enhanced in the early stage of thermal injury. This effect may play an important role in the pathophysiological events of microcirculation and blood–tissue exchange in burns.

Protein kinase C inhibition improves ventricular function after thermal trauma.

To examine the effect of protein kinase C (PKC) inhibition on cardiac performance and intracellular Ca2+ homeostasis. Previous studies have shown that trauma impairs cardiac mechanical function, and recent studies suggest that PKC activation and subsequent perturbations in Ca2+ sequestration/release contribute to this cardiac dysfunction. In this study, anesthetized guinea pigs were given third-degree scald burns over 43 +/- 1% of the total body surface area and resuscitated with lactated Ringer's solution (LR) 4 mL/kg per percent of burn, Parkland formula. Animals with sham burns served as controls (n = 18). Burns were randomly divided into two groups: LR alone (N = 18) or LR + PKC inhibitor, calphostin C (0.1 mg/kg, intravenous bolus), given 30 minutes and 3, 6, and 21 hours after burn (n = 18). Cardiac function was assessed by Langendorff preparation 24 hours after burn in 8 to 12 animals per group. Intracellular calcium concentration ([Ca2+]i) was measured in cardiac myocytes (collagenase digestion) from additional animals in each experimental group (n = 5-9 per group) after Fura-2 AM loading of myocytes; fluorescence ratios were measured with a Hitachi spectrofluorometer. Cardiac dysfunction occurred 24 hours after burn in LR burns as indicated by lower left ventricular pressure and a reduced rate of left ventricular pressure rise and fall, +/-dP/dt (61 +/- 3 mm Hg, 1,109 +/- 44 mm Hg/s, and 880 +/- 40 mm Hg/s, respectively) compared with values measured in sham-burned animals (86 +/- 2 mm Hg, 1365 +/- 43 mm Hg/s, and 1183 +/- 30 mm Hg/s, respectively; p < 0.05). Ventricular function curves confirmed significant postburn contractile depression despite aggressive fluid resuscitation. Cardiac injury in burned animals was indicated by an increase in perfusate creatine kinase and lactate dehydrogenase, and Ca2+ dyshomeostasis was confirmed by increased myocyte [Ca2+]i (sham 151 +/- 6 vs. burn 307 +/- 20 nmol/L, p < 0.05). PKC inhibition improved all indices of cardiac performance, producing left ventricular pressure (82 +/- 3 mm Hg), +/-dP/dt (1,441 +/- 48 and 1,294 +/- 32 mm Hg/s), and left ventricular function curves that were comparable with those of sham-burned animals. In addition, [Ca2+]i in calphostin-treated burned animals (154 +/- 11 nmol/L) was identical to values in sham-burned animals. Our data suggest that PKC may serve as a final common pathway in signal transduction events mediating postburn cardiac dysfunction.

Dosage and Timing of Anti-TNF-α Antibody Treatment Determine Its Effect on Resistance to Sepsis after Injury

Antibody against tumor necrosis factor-α (TNF-α) has improved survival in certain models of sepsis, but it remains unproven in clinical studies. In most of the successful animal studies, efficacy has been shown in previously healthy animals subjected to a septic challenge. Patients at risk for sepsis, however, may be ill for some time before the sepsis supervenes. This situation has been described as a “two-hit” model of critical illness. We have developed an animal burn–sepsis model which conforms to this “two-hit” concept. We have quantified macrophage TNF-α production at different times after the burn (first “hit”) and determined the effect of neutralizing antibody against TNF-α during this period on survival after subsequent sepsis (second “hit”). The objective of this study was to determine the role of TNF-α and the effect of neutralizing antibody against TNF-α in a burn–sepsis model. Animals were subjected to a full thickness burn or sham burn.In vitroTNF-α production from cultured lipopolysaccharide-stimulated splenic adherent cells was determined at various time points thereafter by enzyme-linked immunosorbent assay. Separate animals were treated with neutralizing antibody against TNF-α at different time points after the thermal injury, and survival was determined after septic challenge (cecal ligation and puncture) on Day 10 after the burn. TNF-α production from adherent splenocytes was not elevated in the early days after thermal injury, but was significantly enhanced from Day 6 onward compared with sham-burned animals. Nine percent of the burned mice survived septic challenge compared with 69% of the sham-burned control mice (P< 0.001). Therapy with anti-TNF antibody at 1 × 104neutralizing units (n.u.) kg−1markedly improved outcome if given when TNF-α production was elevated at Day 7 after the burn (survival, 36%;P= 0.01) but did not improve survival when administered at Days 0 or 4 or at the time of the septic challenge (Day 10). High doses of antibody (3.2 × 105n.u.kg−1) were not beneficial and may have been detrimental. These results show that neutralizing antibody against TNF-α may reduce the susceptibility to infection seen after thermal injury, but the timing of administration of the antibody and the dose of antibody used are critical to the outcome. This should be considered when neutralizing antibody against TNF is used in the clinical setting.

Burn Injury Alters Coronary Endothelial Function

This study examined the effects of burn injury on coronary endothelial function and coronary vascular reactivity. Adult rabbits were given a scald burn over 30% of the total body surface area (or sham burn for controls) and resuscitated with Ringer's lactate solution (4 ml/kg/% burn). Subgroups of burned (n= 6) and sham-burned (n= 6) animals were sacrificed at 2, 6, and 24 hr after injury; hearts were harvested and perfused. Changes in coronary perfusion pressure (CPP, mmHg) and coronary vascular resistance (CVR, mmHg/min) were determined at a constant preload and constant coronary flow rate. Changes in coronary endothelial function were determined by the ability of the endothelium to release cGMP as an indicator of nitric oxide production. Compared to values measured in sham burns, CPP and CVR progressively fell during the early postburn period but increased toward values measured in the sham burn group by 24 hr. Cyclic GMP, fmole/ml of coronary perfusate, was significantly lower in burned hearts (27 ± 1) compared to values measured in effluents from sham burn hearts (310 ± 40,P< 0.05). Alterations in coronary effluent cGMP levels after burn injury suggest that thermal injury disrupts coronary endothelial function, likely contributing to postburn changes in cardiac performance.

Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection.

Patients with serious traumatic injury and major burns and an animal model of burn injury were studied to determine the effect of injury on the production of cytokines typical of the T helper-2 lymphocyte phenotype as opposed to the T helper-1 phenotype and on the production of interleukin-12. Perturbations of natural and adoptive immunity are related to the increased susceptibility to infection manifested by seriously injured and burn patients. Earlier work has shown that impaired adoptive immunity after injury is characterized by diminished production of interleukin-2 (IL-2), a product of Th lymphocytes. Exposure of naive Th cells to certain antigens and cytokines causes conversion to either the Th-1 or the Th-2 phenotype. Th-1 cells produce IL-2 and interferon-gamma (IFN-tau) and initiate cellular immunity. Th-2 cells secrete interleukin-4 (IL-4) and interleukin-10 (IL-10) and stimulate production of certain antibodies. Conversion to the Th-1 phenotype is facilitated by IL-12, and conversion to the Th-2 phenotype is promoted by IL-4. The authors believed that serious injury might cause conversion of Th cells to the Th-2 as opposed to the Th-1 phenotype rather than generalized Th suppression. The authors studied circulating peripheral blood mononuclear cells (PBMC) from 16 major burn and 8 trauma patients on 32 occasions early after injury and from 13 age- and sex-matched healthy individuals for cytokine production after phytohemagglutinin stimulation. Also studied was a mouse model of 20% burn injury known to mimic the immune abnormalities seen in humans with burns. Splenocytes from burn mice, 10 to 12 per group, were studied after activation by concanavalin A or by the bacterial antigen Staphylococcus aureus Cowan strain I for cytokine production and cytokine messenger RNA expression as determined by reverse transcriptase polymerase chain reaction. Burn mice were compared with sham-burn controls and attention was focused on day 10 after burn injury, a time when IL-2 production and resistance to infection are highly suppressed. Finally, burn and sham-burn animals, 20 per group, were treated in vivo with IL-12 (25 ng daily for 5 days) and observed for mortality after septic challenge (cecal ligation and puncture [CLP]) performed on day 10 after injury. Peripheral blood mononuclear cells from burn and trauma patients produced less IFN-tau, the index cytokine of Th-1 cells, than PBMCs from healthy individuals 1 to 14 days after burn injury (SE = 77.6 +/- 16 pg/mL patients vs. 141.3 +/- 35 pg/mL controls, p < 0.05). However, production of IL-4, the index cytokine of Th-2 cells, by patient PBMCs was increased (51.0 +/- 13.0 pg/mL patients vs. 26.9 +/- 2.5 controls, p < 0.05). Splenocytes from mice 10 days after burn injury, when compared with sham-burn controls, showed diminished production of IL-2 (1.04 +/- 0.91 units/mL burns vs. 5.8 +/- 0.55 units/mL controls, p < 0.05) and IFN-tau (1.05 +/- 0.7 units/mL burns vs. 12.0 +/- 8.9 units/mL controls, p < 0.05). However, burn splenocytes produced more IL-4 (2492 +/- 157.0 pg/mL burns vs. 672.0 +/- 22.7 pg/mL controls, p < 0.01) and IL-10 (695.2 +/- 20.8 pg/mL burns vs. 567.0 +/- 16.7 pg/mL controls, p < 0.05). Splenocyte production of IL-12 was also reduced after burn (0.20 +/- 0.035 units/mL) as compared with sham burn (0.46 +/- 0.08 units/mL, p < 0.05). The reduction in IL-2, IFN-tau, and IL-12 production by burn splenocytes was reflected by a tenfold decrease in expression of their respective cytokine mRNAs. In vivo IL-12 treatment of burn animals decreased mortality from CLP on day 10 after injury from 85% to 15% (sham-burn mortality after CLP, 15%, p < 0.05) and increased splenocyte IFN-tau production to supranormal levels. Serious injury induced diminished production of IL-1 2 and a shift to the Th-2 phenotype with increased production of IL-4 and IL-10, cytokines known to inhibit Th-1 function. The ability of exogenous IL-12 to restore Th-1 cytokine production and resistance to infection suggests a therapeutic role for IL-12 in the immune dysfunction seen after major injury.

Effects of hypertonic saline and dextran 70 on cardiac functions after burns

The effects of hypertonic saline-dextran (HSD) on cardiac contractility and hemodynamics after burns were studied in anesthetized animals with full-thickness 50% total body surface area burns that were resuscitated with HSD or lactated Ringer solution (LR) alone. No significant difference in cardiac contractility during 6 h postburn was observed between the two groups, as assessed by the end-systolic pressure-volume relationship and the stroke work-end-diastolic volume relationship. An additional bolus of HSD at 6 h postburn caused no significant changes in the end-systolic pressure-volume relationship and stroke work-end-diastolic volume relationship in the burned and sham-burned animals, both of which were resuscitated with HSD. Ten minutes of hemodynamic changes following HSD infusion at 30 min postburn revealed a sudden increase in stroke volume with biphasic responses in left ventricular systolic pressure, which first decreased, then increased, and finally returned to the pre-HSD value. End-diastolic volume was maintained at approximately 110% of the pre-HSD value during this period. We concluded that HSD does not enhance cardiac contractility after severe burns but does produce direct effects on postburn circulation to reduce afterload and augment preload, resulting in a short-lived increase in cardiac output.

BACTERIAL TRANSLOCATION AFTER BURN INJURY

Bacterial translocation (BT) has been shown to occur in stress and trauma. In this study, we examined the contribution of altered intestinal permeability (measured with 51Cr-EDTA) and intestinal blood flow (radioactive microspheres) to the loss of intestinal barrier function after burn injury. A 42.6 +/- 0.6% total body surface area scald burn was produced in Sprague-Dawley rats; sham burn animals served as controls. Rats (29 burn and 20 sham burn) were sacrificed 24 h postburn, and mesenteric nodes (MLN), cecum, spleen, and liver were cultured. The incidence of BT was significantly higher in burn compared to shams (MLN, 55 vs. 15%; spleen, 31 vs. 10%; liver, 31 vs. 10%; p < .05). Cecal concentration of Gram-negative bacteria were similar in all rats (5.2 +/- 0.2 x 10(8) colony forming units/g). 5 h postburn, intestinal blood flow decreased significantly in burn (1.60 +/- .20) compared to shams (2.49 +/- .24 ml/min/g, p = .01); at this time, plasma to luminal clearance of 51Cr-EDTA was greater in the burn (.146 +/- .033, N = 9) than in shams (.050 +/- .010 ml/min/100 g, N = 9, p = .001); 24 h postburn, there was no significant difference in intestinal blood flow in sham (2.86 +/- .46, N = 10) and burn rats (2.29 +/- .44 ml/min/g, N = 11); plasma to intestinal lumen clearance of 51Cr-EDTA was similar in sham (.054 +/- .010, N = 11) and burn rats (.051 +/- .006 ml/min/100 g, N = 12) 24 h postburn.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppressor T-cell levels are unreliable indicators of the impaired immune response following thermal injury.

The presence of increased levels of suppressor T cells after thermal injury and their relevance remain controversial. It is unclear whether suppressor T cells are the cause or result of sepsis complicating thermal injury. Spleen cells from a standardized murine burn model and sham burn controls were studied and the relationship between the levels of suppressor cytotoxic T cells (CD8, Lyt-2+), helper T cells (CD4, L3T4+), response to concanavalin A (ConA) and to phytohemagglutinin (PHA) and interleukin-2 (IL-2) production was examined. Mortality following infection via cecal ligation and puncture (CLP) of matched controls was also studied. At day 7 postburn, mean ConA (70 +/- 12% of control) and PHA response (58% +/- 5.2% of controls) and IL-2 production (43% +/- 5.4%) were significantly less than sham burn values (100%; p less than 0.05). However, the mean percentage of cells staining with anti-Lyt-2 and anti-L3T4 (9.1 +/- 0.59 and 13.9 +/- 0.65) was similar to the mean percentage in sham burn animals (9.4 +/- 0.65 and 16.6 +/- 1.1). Furthermore, no significant differences were observed between burned mice and controls in helper (17.3% +/- 1.8% burn vs. 21.2% +/- 1.7% sham) or suppressor cell levels (7.8% +/- 1.2% burn vs. 8.6% +/- 0.7% sham) or helper-suppressor ratios on day 10 postburn. Mortality of 20 litter-matched controls subjected to CLP on day 10 postburn was 90%, which was significantly greater than the sham burn mortality of 20%.(ABSTRACT TRUNCATED AT 250 WORDS)